Nonclinical Characterization of Bexmarilimab, a Clever-1-Targeting Antibody for Supporting Immune Defense Against Cancers.

Common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) is a multifunctional type-1 transmembrane protein that plays an important role in immunosuppression against tumors. Clever-1 is highly expressed in a subset of human tumor-associated macrophages and associated with poor survival. In mice, Clever-1 supports tumor growth and metastasis formation, and its deficiency or blockage induces T-cell-dependent killing of cancer cells. Therefore, targeting Clever-1 could lead to T-cell activation and restoration of immune response also in patients with cancer. This is studied in an on-going clinical trial [Macrophage Antibody To INhibit immune Suppression (MATINS); NCT03733990] in patients with advanced solid tumors where bexmarilimab, a humanized IgG4 antibody against human Clever-1, shows promising safety and efficacy. Here, we report the humanization and nonclinical characterization of physicochemical properties, biological potency, and safety profile of bexmarilimab. Bexmarilimab showed high affinity to Clever-1 on KG-1 cells and bound to Clever-1 on the surface of classical and intermediate monocytes derived from healthy human blood. Bexmarilimab inhibited the internalization of its natural ligand acetylated low-density lipoprotein into KG-1 cells and increased TNFα secretion from macrophages but did not impair phagocytic clearance. Bexmarilimab did not induce significant cytokine release in human whole-blood cultures, did not contain nonsafe immunogenic glycans, or show any significant binding to human Fcγ receptors or complement pathway component C1q. In vivo, bexmarilimab showed dose-dependent duration of monocyte Clever-1 receptor occupancy in cynomolgus monkeys but did not induce a cytokine storm up to a dose of 100 mg/kg. In conclusion, these data support the clinical development of bexmarilimab for the restoration of immune response in cancers.

Effect of Intravenous Interferon ß-1a on Death and Days Free From Mechanical Ventilation Among Patients With Moderate to Severe Acute Respiratory Distress Syndrome: A Randomized Clinical Trial.

Importance: Acute respiratory distress syndrome (ARDS) is associated with high mortality. Interferon (IFN) ß-1a may prevent the underlying event of vascular leakage. Objective: To determine the efficacy and adverse events of IFN-ß-1a in patients with moderate to severe ARDS. Design, Setting, and Participants: Multicenter, randomized, double-blind, parallel-group trial conducted at 74 intensive care units in 8 European countries (December 2015-December 2017) that included 301 adults with moderate to severe ARDS according to the Berlin definition. The radiological and partial pressure of oxygen, arterial (Pao2)/fraction of inspired oxygen (Fio2) criteria for ARDS had to be met within a 24-hour period, and the administration of the first dose of the study drug had to occur within 48 hours of the diagnosis of ARDS. The last patient visit was on March 6, 2018. Interventions: Patients were randomized to receive an intravenous injection of 10 µg of IFN-ß-1a (144 patients) or placebo (152 patients) once daily for 6 days. Main Outcomes and Measures: The primary outcome was a score combining death and number of ventilator-free days at day 28 (score ranged from -1 for death to 27 if the patient was off ventilator on the first day). There were 16 secondary outcomes, including 28-day mortality, which were tested hierarchically to control type I error. Results: Among 301 patients who were randomized (mean age, 58 years; 103 women [34.2%]), 296 (98.3%) completed the trial and were included in the primary analysis. At 28 days, the median composite score of death and number of ventilator-free days at day 28 was 10 days (interquartile range, -1 to 20) in the IFN-ß-1a group and 8.5 days (interquartile range, 0 to 20) in the placebo group (P = .82). There was no significant difference in 28-day mortality between the IFN-ß-1a vs placebo groups (26.4% vs 23.0%; difference, 3.4% [95% CI, -8.1% to 14.8%]; P = .53). Seventy-four patients (25.0%) experienced adverse events considered to be related to treatment during the study (41 patients [28.5%] in the IFN-ß-1a group and 33 [21.7%] in the placebo group). Conclusions and Relevance: Among adults with moderate or severe ARDS, intravenous IFN-ß-1a administered for 6 days, compared with placebo, resulted in no significant difference in a composite score that included death and number of ventilator-free days over 28 days. These results do not support the use of IFN-ß-1a in the management of ARDS. Trial Registration: ClinicalTrials.gov Identifier: NCT02622724.

Comparison of the efficacy and safety of FP-1201-lyo (intravenously administered recombinant human interferon beta-1a) and placebo in the treatment of patients with moderate or severe acute respiratory distress syndrome: study protocol for a randomized controlled trial.

BACKGROUND: Acute respiratory distress syndrome (ARDS) results in vascular leakage, inflammation and respiratory failure. There are currently no approved pharmacological treatments for ARDS and standard of care involves treatment of the underlying cause, and supportive care. The vascular leakage may be related to reduced concentrations of local adenosine, which is involved in maintaining endothelial barrier function. Interferon (IFN) beta-1a up-regulates the cell surface ecto-5'-nucleotidase cluster of differentiation 73 (CD73), which increases adenosine levels, and IFN beta-1 may, therefore, be a potential treatment for ARDS. In a phase I/II, open-label study in 37 patients with acute lung injury (ALI)/ARDS, recombinant human IFN beta-1a was well tolerated and mortality rates were significantly lower in treated than in control patients. METHODS/DESIGN: In this phase III, double-blind, randomized, parallel-group trial, the efficacy and safety of recombinant human IFN beta-1a (FP-1201-lyo) will be compared with placebo in adult patients with ARDS. Patients will be randomly assigned to receive 10 µg FP-1201-lyo or placebo administered intravenously once daily for 6 days and will be monitored for 28 days or until discharged from the intensive care unit. Follow-up visits will then take place at days 90, 180 and 360. The primary endpoint is a composite endpoint including any cause of death at 28 days and days free of mechanical ventilation within 28 days among survivors. Secondary endpoints include: all-cause mortality at 28, 90, 180 and 360 days; organ failure-free days; length of hospital stay; pharmacodynamic assessment including measurement of myxovirus resistance protein A concentrations; and measures of quality of life, respiratory and neurological function at 180 and 360 days. The estimated sample size to demonstrate a reduction in the primary outcome between groups from 30% to 15% is 300 patients, and the study will be conducted in 70-80 centers in nine countries across Europe. DISCUSSION: There are no effective specific treatments for patients with ARDS and mortality rates remain high. The results from this study will provide evidence regarding the efficacy of a potential new therapeutic agent, FP-1201-lyo, in improving the clinical course and outcome for patients with moderate/severe ARDS. TRIAL REGISTRATION: European Union Clinical Trials Register, no: 2014-005260-15 . Registered on 15 July 2017.

The effect of intravenous interferon-beta-1a (FP-1201) on lung CD73 expression and on acute respiratory distress syndrome mortality: an open-label study.

BACKGROUND: Pulmonary vascular leakage occurs early in acute respiratory distress syndrome (ARDS). Mortality is high (35-45%), but no effective pharmacotherapy exists. Production of anti-inflammatory adenosine by ecto-5'-nucleotidase (CD73) helps maintain endothelial barrier function. We tested whether interferon-beta-1a (IFN-beta-1a), which increases CD73 synthesis, can reduce vascular leakage and mortality in patients with ARDS. METHODS: In ex-vivo studies, we first established that IFN-beta-1a induced CD73 up-regulation in cultured human lung tissue samples. We then tested the safety, tolerability, and efficacy of intravenous human recombinant IFN-beta-1a (FP-1201) in patients with ARDS in an open-label study (comprising dose-escalation and expansion phases). We recruited patients from eight intensive care units in the UK. Eligible patients were aged 18 years or older, had ARDS, and were being treated with assisted ventilation. We established an optimal tolerated dose (OTD) in the first, dose-escalation phase. Once established, we gave all subsequently enrolled patients the OTD of intravenous FP-1201 for 6 days. We assessed 28-day mortality (our primary endpoint) in all patients receiving the OTD versus 28-day mortality in a group of patients who did not receive treatment (this control group comprised patients in the study but who did not receive treatment because they were screened during the safety windows after dose escalation). This trial is registered with ClinicalTrials.gov, number NCT00789685, and the EU Clinical Trials Register EudraCT, number 2008-000140-13. FINDINGS: IFN-beta-1a increased the number of CD73-positive vessels in lung culture by four times on day 1 (p=0·04) and by 14·3 times by day 4 (p=0·004). For the clinical trial, between Feb 23, 2009, and April 7, 2011, we identified 150 patients, of whom 37 were enrolled into the trial and given treatment. The control group consisted of 59 patients who were recruited to take part in the study, but who did not receive treatment. Demographic characteristics and severity of illness did not differ between treatment and control groups. The optimal tolerated FP-1201 dose was 10 µg per day for 6 days. By day 28, 3 (8%) of 37 patients in the treatment cohort and 19 (32%) of 59 patients in the control cohort had died-thus, treatment with FP-1201 was associated with an 81% reduction in odds of 28-day mortality (odds ratio 0·19 [95% CI 0·03-0·72]; p=0·01). INTERPRETATION: FP-1201 up-regulates human lung CD73 expression, and is associated with a reduction in 28-day mortality in patients with ARDS. Our findings need to be substantiated in large, prospective randomised trials, but suggest that FP-1201 could be the first effective, mechanistically targeted, disease-specific pharmacotherapy for patients with ARDS.

Altered expression of syndecan-1 in prostate cancer.

Syndecan-1 is a cell surface heparan sulfate proteoglycan expressed by epithelial cells. It interacts with growth factors, matrix components, and other extracellular proteins, and is thought to be involved in processes such as cell growth, differentiation and adhesion. The expression of syndecan-1 appears generally downregulated in human carcinomas and in experimental cancer models, whereas transfectional expression of syndecan-1 in cultured cancer cells has been shown to inhibit their growth and other aspects of malignant behavior. These findings suggest that analysis of syndecan-1 expression might be of prognostic value in cancer diagnosis, and studies on some carcinomas indeed point to an inverse correlation between syndecan-1 expression and cancer prognosis. So far, little information has been available on the expression of syndecan-1 in human prostate and prostate disease. We have generated and characterized novel antibodies against syndecan-1 and applied them to immunohistochemical staining of specimens representing normal prostate as well as benign and malignant (n=23) prostate disease. The results indicate that syndecan-1 expression is altered but not uniformly absent in prostate cancer, which is in contrast to the expression of high-molecular-weight cytokeratins. The data initially suggest an inverse correlation between syndecan-1 expression and Gleason grade of the tumor, and warrant a larger study to assess the potential prognostic value of analysing syndecan-1 expression in prostate carcinoma.

Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling.

Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling.

Protein kinase A balances the growth factor-induced Ras/ERK signaling.

Protein kinase A (PKA) has been proposed to regulate the signal transduction through the Ras/extracellular-regulated kinase (ERK) pathway. Here we demonstrate that when the PKA activity was inhibited prior to growth factor stimulus the signal flow through the Ras/ERK pathway was significantly increased. Furthermore, the data indicated that this PKA-mediated regulation was simultaneously targeted to the upstream kinase Raf-1 and to the ERK-specific phosphatase mitogen-activated protein kinase phosphatase-1 (MKP-1). Moreover, our data suggested that the level of PKA activity determined the transcription rate of mkp-1 gene, whereas the Ras/ERK signal was required to protect the MKP-1 protein against degradation. These results point to a tight regulatory relationship between PKA and the growth factor signaling, and further suggest an important role for basal PKA activity in such regulation. We propose that PKA adjusts the activity of the Ras/ERK pathway and maintains it within a physiologically appropriate level.

Cooperation of protein kinase A and Ras/ERK signaling pathways is required for AP-1-mediated activation of fibroblast growth factor-inducible response element (FiRE).

Recent studies suggest a crucial role for protein kinase A (PKA) in the regulation of growth factor signaling. However, the effect of PKA on the transcription of growth factor-responsive genes has drawn far less attention. Here we have investigated the signaling mechanisms involved in the activation of an activator protein-1 (AP-1)-driven, growth factor-specific enhancer element, fibroblast growth factor-inducible response element (FiRE). The activation was found to be mediated by three phorbol 12-O-tetradecanoate-13-acetate-response element-related DNA elements of FiRE, including motif 4 and two distinct elements of motif 5 (referred to as M5-1 and M5-2). All three elements were required for full FiRE activity. Stimulation of cells with fibroblast growth factor-2 (FGF-2) induced the binding of AP-1 to motif 4 and M5-2, whereas M5-1 did not show detectable binding. The FGF-2-induced FiRE activation appeared to require cooperational function of the Ras/ERK and PKA pathways. Inhibition of either of the pathways abolished the binding of AP-1 complexes to motif 4 and motif 5 and the subsequent FiRE activation. By contrast, costimulation of cells with FGF-2 and the PKA activator 8-bromo-cyclic AMP increased the binding of AP-1 to FiRE and potentiated the level of transcriptional activity. The cooperational function of these two pathways was confirmed by experiments with cell lines stably expressing 4-hydroxytamoxifen-inducible oncogenic Raf-1 (DeltaRaf-1:ER[DD]). Noticeably, the induction systems showed variations with respect to regulation of AP-1-driven activation of FiRE. These differences were likely to originate from the ability of these two systems to induce the differential activation pattern of the Ras/ERK pathway.

Expression and characterization of minican, a recombinant syndecan-1 with extensively truncated core protein.

Syndecan-1 is an integral membrane heparan sulfate/chondroitin sulfate proteoglycan, involved in the control of cell growth and differentiation. The biological activities of syndecan-1 involve interactions with a variety of extracellular ligands, such as growth factors and matrix components, that are mainly mediated by the heparan sulfate moieties. The expression of syndecan-1 is downregulated in various malignant tumors, and low levels of expression appear to correlate with poor prognosis of some cancer types. On the other hand, the extracellular portion of syndecan-1 (ectodomain) has been demonstrated to inhibit the proliferation of various cancer cells in culture, suggesting that proteoglycan-like molecules should be studied further with regard to their antitumor activities. We have expressed, in CHO cells, a truncated syndecan-1 ectodomain ("minican") harboring domains for glycosaminoglycan attachment and antibody recognition. Analysis of recombinant minican indicates that it shares some of the biochemical and biological characteristics attributed to syndecan-1 ectodomain. Minican was thus substituted with heparan sulfate chains and bound to extracellular matrix proteins as well as fibroblast growth factors. Notably, minican inhibited the proliferation of S115 mouse mammary carcinoma cells and the effect seemed to involve inhibition of the Ras/Erk signaling pathway. Our data suggest that recombinant syndecan-1 with a minimal protein component is biologically active. This information may provide useful in further design of proteoglycan-like antitumor molecules.